Human Reproduction vol.15 no.12 pp.2563–2566, 2000
CASE REPORT
Do morphological anomalies reflect chromosomal aneuploidies?*
Ste´phane Viville1,3, Richard Mollard2, Marie-Lorraine Bach1, Ce´dric Falquet1, Pierre Gerlinger1 and Ste´phanie Warter1 1Service
de Biologie de la Reproduction SIHCUS-CMCO, 19, rue Louis Pasteur BP120, 67303 Schiltigheim, France and 2Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/ INSERM/ULP, 1, rue Laurent Fries, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France 3To
whom correspondence should be addressed. E-mail:
[email protected]
In cases of severe teratozoospermia, the current morphological criteria used to assess chromosomal status is insufficient for the selection of spermatozoa for intracytoplasmic sperm injection (ICSI). Case histories are reported of four patients presenting 100% teratozoospermia, and the integrity of their individual chromosomal statuses is determined using a three-colour fluorescence in-situ hybridization (FISH) technique. Patient 1 presented shortened flagella syndrome, patient 2 globozoospermia, patient 3 spermatozoa with irregular acrosomes, and patient 4 macrocephalic spermatozoa with associated multiple flagella. Three-colour FISH analysis using chromosome X, Y and 1 -specific probes showed that ~95% of the spermatozoa analysed from patients 1, 2 and 3 presented X,1 and Y,1 signals, X,Y ratios and aneuploidy/ diploidy rates comparable with those observed in normal controls. In contrast, patient 4 showed a highly elevated Y to X sex ratio and a highly elevated aneuploidy/diploidy rate. Three-colour FISH analysis thus demonstrates an increased incidence of chromosomal abnormalities in association with macrocephalic spermatozoa. Moreover, the analysis shows that in patients affected with either globozoospermia, shortened flagella syndrome or a condition of abnormal acrosomal spermatozoa, no association exists between chromosomal status and phenotype. Since these patients display normal haploid, sex chromosome and aneuploidy status, ICSI can be conceivably offered as a treatment for their infertility. Key words: chromosomal aneuploidy/ICSI/infertility/morphological anomaly/teratozoospermia
*The results of these studies have been presented in part at the 16th Annual Meeting of the ESHRE, Bologna, Italy, 2000. © European Society of Human Reproduction and Embryology
Introduction Intracytoplasmic sperm injection (ICSI) is currently offered as a treatment for severe male infertility. However, one major question raised by the use of ICSI relates to the possibility of transmitting genetic abnormalities to any resulting conceptus arising from limitations inherent in the morphological criteria currently used to select spermatozoa for fertilization (Bonduelle et al., 1999; Lamb, 1999; Schlegel, 1999). Deviations from these morphological parameters are considered to contraindicate the use of ICSI due to the suggestion that spermatozoa from infertile men affected with oligoasthenoteratozoospermia may present an increased frequency of meiotic and cytogenetic abnormalities (Pieters et al., 1998). More recent data, however, have shown that neither sperm count nor morphology influence ICSI outcome, thus leading to the hypothesis that these parameters do not necessarily reflect the chromosomal status of affected spermatozoa (Nagy et al., 1995). As demonstrated previously, therefore, the possibility remains that ICSI can be used successfully to treat male infertility even when it is not possible to locate morphologically normal spermatozoa (Tasdemir et al., 1997). The case histories are reported of four patients presenting four different predominant types of anomalies associated with absolute teratozoospermia for which a three-colour fluorescence in-situ hybridization (FISH) analysis was performed. It is intended to demonstrate that aberrant morphology does not necessarily indicate abnormal chromosomal status, and to discuss the associated likelihood of a genetic risk to any potential conceptus of these patients, based on their specific presentation of teratozoospermia. Materials and methods For each patient, at least two consecutive semen samples showed severe oligoasthenoteratozoospermia, with 100% morphologically abnormal spermatozoa, according to the criteria of the World Health Organization (WHO, 1992). Each patient presented a different predominant morphological anomaly of their semen (Table I). For patient 1, 54% of the spermatozoa displayed shortened flagella syndrome; for patient 2, all spermatozoa showed globozoospermia; for patient 3, 96% of spermatozoa showed abnormal acrosomes; and for patient 4, 64% of spermatozoa showed a macrocephalic sperm head associated with multiple flagella. All four patients presented a normal X,Y karyotype as examined on peripheral lymphocytes, and a normal concentration range of testosterone and FSH. Patient 2 had an infertile brother presenting the same semen phenotype, thus suggesting a genetic trait. With the exception of patient 3, where the female partner presented a unilateral tubal obstruction, all other female partners did not present any obvious infertility factor. Patient 1 had undertaken a
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Table I. Patient clinical history and ICSI outcomes Patient Age Female Sperm no. (years) partner’s conc. age (years) (⫻106/ml)
Type of teratospermia
ICSI attempts
No. of oocytes and MII
No. of day 2 embryos
No. of Biochemical Clinical Spontaneous transferred pregnancies pregnancies abortions embryos (n) (n) (n)
1
31
36
7
34% absence of flagella 54% shortened flagella
2
25
24
2
100% globozoospermia
3
36
35
4
96% abnormal acrosome
4
48
32
9
64% macrocephalic 52% abnormal acrosome 28% multiple flagella
1 2 3 4 1 2 1 2 1 2 3
26/20 18/13 0 12/8 11/6 6/6 5/5 6/4 12/11 10/2 8/4
11 8 0 7 0 0 5 3 5 1 1
3 3 0 3 0 0 2 2 3 1 1
0 1 0 1 0 0 0 1 0 0 0
0 0 0 1 0 0 0 1 0 0 0
0 0 0 1 0 0 0 1 0 0 0
MII ⫽ metaphase II oocytes.
total of four ICSI attempts which had resulted in three embryo transfers giving rise to two clinical pregnancies, both of which spontaneously aborted before the end of the third month. Patient 2 had undertaken two attempts with no fecundity. Patient 3 had undertaken a total of two attempts, both of which resulted in embryo transfer, one giving a clinical pregnancy which aborted spontaneously before the end of the first trimester. Patient 4 had undertaken three ICSI cycles with two embryo transfers, but no pregnancies (Table I). In order to establish the risk for these patients of injecting a chromosomally abnormal spermatozoon during the ICSI procedure, and to determine their chances of conceiving with their own semen, a three-colour FISH analysis was performed, using probes specific for chromosomes X, Y and 1. The use of two sex chromosomal probes and an autosomal-specific probe allowed determination of the ploidy status of each spermatozoon analysed. Probes included satellite probes for chromosome X (pBam X5) and 1 (pUC1.77), and a probe recognising the heterochromatic region of chromosome Y (pCY98). Probes were directly labelled using a nick translation kit (Boehringer, Mannheim, Germany) with: (i) rhodamine-4-dUTP (Amersham, Les Ullis, France) for the chromosome X probe; (ii) fluorescein isothiocyanate (FITC)-12-dUTP (Boehringer) for the chromosome Y probe; and (iii) a mixture (1:1) of FITC-12dUTP and rhodamine-4-dUTP for the chromosome 1 probe, as described previously (Viville et al., 1998). In order to study spermatozoa used for ICSI, FISH analysis was performed on one semen specimen from each patient after the spermatozoa had been purified through a three-layer Percoll gradient. Sperm nuclei decondensation was obtained by a 4 min incubation in 0.1 mol/l Tris–HCl buffer (pH 8) containing 25 mmol/l dithiothreitol (DTT). Hybridization was performed in 60% formamide/2⫻ sodium chloride/sodium citrate (SSC)/10% dextran sulphate for 2 h following a 1 min denaturation at 75°C. Post-hybridization washes included 5 min in 60% formamide/2⫻ SSC and 5 min in 2⫻ SSC at 42°C, followed by two 5 min washes in 4⫻ SSC/0.05% Tween 20 at room temperature. After dehydration through an ethanol series, slides were counterstained with 4⬘,6-diaminidino-2-phenylindol (DAPI) in antifading solution (Vector, Burlingame, CA, USA). Signal analysis was performed on a Zeiss microscope according to published scoring criteria (Hopman et al., 1988). Results were compared with two controls with normal sperm characteristics and proven fertility. Hybridization efficiencies ranged between 92.6 and 99.6%, while interpretable signals ranged between 68.3 and 95.3% (Table II).
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Results Results of the FISH analysis are presented in Table II. For Patients 1, 2 and 3, between 93.1 and 95.3% of the spermatozoa presented a normal X,1 and Y,1 signal, with a sex ratio of 1.03. The aneuploidy/diploidy rate varied between 4.7 and 7.1% compared with a mean of 4.9% in the controls. This higher level of aneuploidy in our controls when compared with previously published data (0.03% to 0.34%; Guttenbach et al., 1997), results from our relatively more stringent criteria for its score. Although nullisomic signals may reflect a hybridization failure, in addition to the traditional consideration of disomic and diploid signals, nullisomic signals were also counted as aneuploid in order to present the maximal risk (and thus information) to the patients. We consider this attitude to offer the most ethical approach. The diploidy rate of patient 1 (0.4%) was significantly greater than that of the two controls (0.1% and 0.02%) (P ⬍ 0.05; see Discussion). Patient 4, who presented 64% macrocephalic spermatozoa, showed the highest aneuploidy rate (66.9%; Table II). In addition, the sex ratio was 4.1 in favour of Y,1, and the diploidy rate was 22.3%. In total, 89.2% of the spermatozoa presented an abnormal chromosomal constitution. For this patient, therefore, only 10.8% of the spermatozoa presented a normal haploid FISH signal for the three chromosomes analysed. Discussion ICSI circumvents natural barriers such as the female genital tract, cells of the corona radiata, the zona pellucida and the cytoplasmic membrane. Questions concerning the safety of injecting a potentially morphologically abnormal spermatozoon have thus been raised (Engel et al., 1996). In order to contribute to this debate, we have performed a three-colour FISH analysis on spermatozoa from four patients presenting four different types of absolute teratozoospermia. Spermocytograms of each patient demonstrated the following predominant morphological abnormalities: shortened flagella syndrome (patient 1), globozoospermia (patient 2), abnormal acrosomes (patient 3) and
Morphological anomalies and chromosomal aneuploidies
Table II. Results of fluorescence in-situ hybridization analyses Patient no.
No. of spermatozoa analysed Hybridization efficiency (%) Interpretable signals (%) FISH signals X1 Y1 _, X _,Y _, 1 _, XY _, XX _, YY _, 1,1 X,1,1 Y, 1,1 XY,1 XY,1,1 YY, 1,1 XYY,1 XXY,1,1 XYY,1,1 Y,1,1,1 XY,1,1,1 XYY,1,1,1 Others Aneuploidy rate (%) Diploidy rate (%)
Control
1
2
3
4
2253
3716
5000
1656
A
B
7423
5064
99.6
99.1
98.2
92.6
99.4
99.7
77.6
95.3
83.5
68.3
96.8
87.3
46.4 46.7 0 0 4.4 0.7 0.1 0.3 1.1 0 0 0 0.4 0 0 0 0 0 0 0 0 6.5 0.4
47.2 48.1 0 0 2.8 0.4 0.5 0.2 0.9 0 0 0 0.1 0 0 0 0 0 0 0 0 4.6 0.1
45.6 47.3 0 0 4.9 0.1 0.1 0.7 1.1 0 0 0 0.2 0 0 0 0 0 0 0 0 6.9 0.2
2.1 8.7 0 0 0.3 13.4 8 14 31 3.3 14.4 13.4 22.3 2.8 1.8 2.4 3.3 3.7 6.5 2.2 10.5 66.9 22.3
45.2 49.5 0 0 4.5 0.06 0.01 0.1 0.6 0 0 0 0.1 0 0 0 0 0 0 0 0 5.3 0.1
49.3 46.3 0 0 3.5 0.07 0.07 0.3 0.5 0 0 0 0.02 0 0 0 0 0 0 0 0 4.4 0.02
Results are presented as the percentage of sperm cells with a readable hybridization signal.
macrocephalic sperm heads (patient 4). For patients 1, 2 and 3, the aneuploidy/diploidy rate (4.7–7.1%) was comparable with that in fertile controls (4.4–5.4%). The increased diploidy status observed in patient 1 (0.4%) was largely comparable with that found in older patients and other control populations. Thus, we do not consider that, relative to the general population, this should impede the chances of patient 1 to conceive a chromosomally unaffected child, although informative counselling regarding this point is recommended (for reviews, see Downie et al., 1997; Egozcue et al., 2000). In the case of patient 4, ~90% of the spermatozoa analysed were aneuploid or diploid. A sex ratio distortion in favour of the Y-bearing spermatozoa was also found (In’t Veld et al., 1997); no explanation was apparent for this phenomenon. Other workers (Martin and Rademaker, 1988), using the human spermatozoon–hamster oocyte fusion karyotype method, found that there was no significant correlation between the frequencies of chromosomally and morphologically abnormal spermatozoa. Karyotyping of human spermatozoa by injection into mouse oocytes showed that some morphological sperm head abnormalities are associated with an elevated aneuploidy rate (Lee et al., 1996); however, no increase was seen with macrocephalic spermatozoa. This latter result contradicts the present data and all other known FISH analyses reported on patients with macrocephalic spermatozoa. In these cases, as reported here, virtually all
spermatozoa were chromosomally abnormal, a finding also supported by others (Kahraman et al., 1999), who studied the fertility and ICSI outcome of 17 patients with macrocephalic spermatozoa. Although chromosomal content was not assessed by the latter group, the observed low fertility rate may be explained by a high frequency of sperm aneuploidy. In addition to the case of macrocephaly, the chromosomal content of spermatozoa from three patients affected with other types of absolute teratozoospermia was studied. To our knowledge, the present study provides the first report of a three-colour FISH analysis of spermatozoa from cases of globozoospermia, shortened flagella syndrome and spermatozoa displaying abnormal acrosomes. The absence of any significant increase in aneuploidy rate suggests that there is no significant relationship between the frequency of numerical chromosomal abnormalities and these specific morphological abnormalities. Our results thus suggest that the chance of conceiving a child with a numerical chromosomal abnormality following ICSI does not appear to be increased for these patients. However, it must be considered reasonable that such causes of infertility may be transmitted to any resulting offspring. For example, globozoospermia has been characterized in mice lacking the casein kinase II α⬘ catalytic subunit (Xu et al., 1999). It appears likely, therefore, that some cases of globozoospermia in humans—as was suggested previously—may represent a genetic trait (Florke-Gerloff et al., 2565
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1984). Even if the chance of conceiving a chromosomally normal child appears reasonable, there remains the genetic risk of transmitting the paternal phenotype to a male child. In such cases ICSI may introduce a new notion in genetics, the ‘heridity of infertility’. In conclusion, on the basis of the results presented here and from other studies, ICSI should not be recommended to patients presenting macrocephalic spermatozoa. For the other cases, additional studies are needed to estimate the genetic risk involved and the chances of conception. Further studies should also be performed to establish specific aneuploidy rates associated with other predominant morphological abnormalities.
Acknowledgement S.Viville and R.Mollard contributed equally to these studies.
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World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK. Xu, X., Toselli, P.A., Russell, L.D. et al. (1999) Globozoospermia in mice lacking the casein kinase II alpha’ catalytic subunit. Nature Genet., 23, 118–21. Received on June 5, 2000; accepted on August 24, 2000
